1. Field of the Invention
The present invention relates generally to transcriptional activator proteins and to methods for altering gene expression, cellular function and metabolism. In particular, this invention concerns sterol regulatory element (SRE) binding proteins (SREBPs) and DNA segments encoding such proteins. Methods for using SREBP-1 to stimulate SRE-1-mediated gene transcription in the presence of sterols are also disclosed, which are contemplated for use in reducing plasma cholesterol levels and in controlling hypercholesterolemia and its associated diseases.
2. Description of the Related Art
There is currently relatively little knowledge concerning feedback suppression mechanisms involved in eukaryotic gene regulation. In animal cells, most attention has focused on positively-regulated systems in which hormones, metabolic inducers, and developmental factors increase transcription of genes. These inducing agents are generally thought to activate or form complexes with proteins that stimulate transcription by binding to short sequences of 10 to 20 base pairs (bp) in the 5'-flanking region of the target gene. Such elements, termed GRE, MRE, and IRE have been reported for glucocorticoid, metal and interferon regulatory elements, respectively (Yamamoto, 1985; Stuart et al., 1984; Goodbourn et al., 1986).
Important advances have been made recently concerning other DNA segments which are capable of conferring control capability to known genes in eukaryotic systems. For example, transcription of the gene for the low density lipoprotein (LDL) receptor is regulated by a 10 base pair (bp) element in the 5' flanking region designated sterol regulatory element-1 (SRE-1; Goldstein and Brown, 1990; U.S. Pat. No. 4,935,363). The receptor provides cholesterol to cells by binding and internalizing LDL, a plasma cholesterol transport protein. When cellular cholesterol demands are high, as when cells are grown in the absence of sterols, this element is transcriptionally active, the cells produce large numbers of LDL receptors and LDL is internalized rapidly. On the other hand, when sterols accumulate within cells, the SRE-1 is silenced, and cells reduce the number of LDL receptors, thereby preventing cholesterol over accumulation. This feedback regulatory system controls not only the cholesterol content of cells, but also that of plasma (Brown and Goldstein, 1986). When hepatic LDL receptors are repressed by intracellular accumulation of dietary cholesterol, LDL is not taken up into the liver at a normal rate, and the lipoprotein builds up to high levels in the blood.
The 10 bp SRE-1 lies within a 16-base pair (bp) sequence, designated Repeat 2, that is 53 bp upstream of the transcription start site of the LDL receptor gene (Smith et al., 1990). This sequence is the central member of a series of three imperfect repeats in the 5' flanking region, all of which are required for high level transcription (Goldstein and Brown, 1990; Smith et al., 1990; Sudhof et al., 1987). Repeats 1 and 3 bind Sp1, a constitutive transcription factor. Mutations in any of the three repeat sequences abolish high-level transcription in sterol-deprived cells (Smith et al., 1990; Sudhof et al., 1987; Dawson et al., 1988).
The activity of Repeats 1 and 3, although necessary, is not sufficient for high level transcription. An additional positive contribution is required from Repeat 2, which does not bind Sp1 (Smith et al., 1990; Sudhof et al., 1987; Dawson et al., 1988). Mutational analysis suggests that Repeat 2 binds a conditionally positive transcription factor that is active only under conditions of sterol deprivation (Smith et al., 1990). When sterols are added to cells, the contribution of Repeat 2 is abolished, and the rate of transcription falls.
The nucleotides within Repeat 2 that are necessary for its transcriptional activity have been delineated partially through in vitro mutagenesis and expression studies in permanently transfected CHO cells. The relevant nucleotides include the SRE-1 10 bp stretch which has the sequence ATCACCCCAC, SEQ ID NO:27 (Smith et al., 1990). The essential elements of this sequence have been shown to be conserved in evolution as far back as the last common ancestor of humans and frogs (Mehta et al., 1991).
Unfortunately, despite the elucidation of the SRE-1 DNA sequence, the nature of the putative transcription factor that binds to SRE-1 remained unknown. Two candidates have been proposed (Rajayashibth et al., 1989; Stark et al., 1992), but the proteins in these reports did not show specific binding which precisely correlated with the transcriptional activity of modified SRE-1 elements, and purification of the putative binding proteins was not reported.
The identification of a protein that binds to the SRE-1 sequence and promotes transcription would be advantageous, particularly if DNA segments encoding such a protein were available so that it could be produced in large quantities. A purified SRE transcriptional activator that could be used to promote SRE-1-mediated gene transcription in the presence of sterols would be even more useful. The availability of such a protein would represent a medical breakthrough as it could be used to promote LDL receptor gene transcription, which is normally downregulated by sterols, and to reduce plasma LDLcholesterol levels.